Methods, procedures and kits for providing maximum depth enrichment sequencing for identification for enrichment of rare genomic variants

ABSTRACT

Exemplary embodiments of methods, kits, systems and computer-accessible medium can be provided for facilitating maximum depth enrichment sequencing for identifying rare genomic variants.

CROSS REFERENCE TO RELATED APPLICATIONS

This application relates to and claims priority from U.S. ProvisionalPatent Application Ser. No. 63/013,927, filed Apr. 22, 2020, thedisclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

The present disclosure is directed to exemplary methods, kits, systemsand computer-accessible medium for providing maximum depth enrichmentsequencing in combination with DNA barcoding for an enrichment of raregenomic variants.

BACKGROUND INFORMATION

There are several existing methods that correct for error ratesintrinsic in library preparation and sequencing, including duplexsequencing and Maximum Depth Sequencing (MDS). These methods usedegenerate molecular tags, otherwise known as “barcodes”, to labelredundant copies of an original genomic molecule. Following sequencing,these barcodes can be used to form a consensus sequence that is robustto polymerase and sequencing errors, decreasing error rates from 1E-2 to1E-6 and below. These advances have given scientists the ability todetect extremely rare genomic variants in a population, increasingsensitivity in cancer prognosis and bacterial metagenomics. However,these techniques all have a major shortcoming in searching for raremutants. Because these mutants are rare, they are by definition a smallproportion of a larger wild-type fraction. To detect a mutant thatexists at a frequency of 1E-6, one million normal sequences need to besequenced to identify a single mutant sequence. Thus, a large amount ofsequencing space is wasted in addition to the excess coverage alreadynecessary for implementing any type of barcoding.

Despite steadily decreasing sequencing costs over the past two decades,detection of rare mutants is still incredibly expensive. A recent studydetecting rare variants in the blood of lung cancer patients estimated acost of $1,750 per region of interest, per patient as of 2017 (SeeTRACERx Consortium et al. Phylogenetic ctDNA analysis depictsearly-stage lung cancer evolution. Nature 545, 446-451 (2017).). This issimply unaffordable in managing patients known to have cancer,especially since multiple samples are collected throughout the diseasecourse and several tumor regions must be examined. It is even lessunrealistic in early screening applications, where a large panel ofhundreds of tumor hot spots need to be surveyed.

Moreover, barcoding techniques can only successfully form consensussequences for a limited number of original genomic molecules. It is onlyreasonable to screen less than half a million cells—costs linearlyincrease with the number of desired genomes—and a normal laboratoryblood draw contains 10 million nucleated cells. Therefore, in mostreasonable use cases, current technologies are only sensitive to 5E-5and can only utilize 5% of the information in a single blood draw.

Clearly there is need for providing methods, systems andcomputer-accessible medium that address at least some of thedeficiencies described herein while preserving the error correctionafforded by barcoding.

EXEMPLARY SUMMARY OF THE DISCLOSURE

Such need is addressed with the exemplary methods, kits, systems andcomputer-accessible medium described herein.

For example, in one exemplary embodiment, a method can be provided forsequencing a nucleic acid, such as a DNA, preferably a genomic DNA, oran RNA, with the method comprising:

-   -   (a) digesting the nucleic acid (i.e., the original molecule),        such a genomic DNA, at the 3′ end of a region of interest to        obtain a digested nucleic acid;    -   (b) performing a linear amplification of the digested nucleic        acid, such as a genomic DNA, with a primary barcoded adapter        comprising a primary barcode to directly barcode the original        molecule, where a regular DNA polymerase may be used in        performing the linear amplification for sequencing DNA, where a        reverse transcriptase may be used in (b) for sequencing a RNA;    -   (c) removing unused primary barcodes by digestion with a        single-stranded DNA exonuclease to obtain a first product;    -   (d) performing N cycles of linear amplification of the first        product using a plurality of secondary barcoded adapters, each        of the plurality of secondary barcoded adapters comprising a        secondary barcode;    -   (e) removing unused secondary barcodes to produce a second        product comprising a first library of multiple copies of the        original molecule, wherein the N cycles of linear amplification        add the secondary barcode such that each primary barcode has N        secondary barcodes;    -   (f) performing a mutant enrichment on the second product to        obtain a second library; and    -   (g) sequencing the second library.

In the above exemplary procedure (a), when the nucleic acid is a DNA,digesting the DNA may be performed by using an enzyme. Examples of theenzyme that can be used to digest the DNA include a restriction enzymeand other types of nucleases such as CRISPR/Cas9.

In the above procedure (a), when the nucleic acid is an RNA, digestingthe RNA may be performed by using an enzyme. Examples of the enzyme thatcan be used to digest the RNA include RNAse H and eukaryotic Argonaute.

The removing unused secondary barcodes may be performed by either byadditional one cycle of PCR with a reverse amplifier to protectsingle-stranded linear amplification products followed by digestion witha single-stranded DNA exonuclease, or by size selection, for example,using beads, gel and/or column purification.

The exemplary method may further utilize an exponential polymerase chainreaction (PCR) of the linear amplification product between the removalof the unused secondary barcodes and the performance of the mutantenrichment.

According to certain exemplary embodiments of the present disclosure,the exemplary method may further include performing an analysis of adata obtained in the above sequencing procedure (g). The data analysismay include, for example, grouping reads by the primary barcode (R) intoa plurality of groups and plurality of groups and considering the numberof primary barcodes R, building consensus, considering the number of thesecondary barcodes (S) and R for each group, and calling for mutants.

In another exemplary embodiment, a method can be provided for sequencingan RNA, the method comprising:

-   -   (a) digesting the RNA at the 3′ end of a region of interest to        obtain a digested nucleic acid;    -   (b) performing a linear amplification of the digested RNA with a        primary barcoded adapter comprising a primary barcode to        directly barcode the original molecule, wherein a reverse        transcriptase may be used in (b);    -   (c) removing unused primary barcodes by digestion with a        single-stranded DNA exonuclease to obtain a product;    -   (d) performing exponential PCR of the product obtained in (c) to        obtain a product; and    -   (e) sequencing the product obtained in (d).

In the above exemplary procedure (a), digesting the RNA can be performedby using an enzyme. Examples of the enzyme that may be used to digestthe RNA include RNAse H and eukaryotic Argonaute.

In further exemplary embodiments of the present disclosure, the methodmay include the following features: (a) a unique implementation ofsecondary barcodes prior to mutant enrichment to protect againstpolymerase errors; (b) a mutant enrichment process; and/or (c) a uniquegeneralizable method for data analysis of double barcoded sequences.

The exemplary mutant enrichment in procedure f) may include:

-   -   1) generating single stranded DNA from the second product    -   2) annealing a reverse-complement wild type sequence that does        not overlap the degenerate regions of the first library to form        homo- or heteroduplexes that correspond the wild-type or mutant        sequences respectively;    -   3) digesting homoduplexes using a duplex specific nuclease to        generate an enriched product    -   4) repetition of 2) and 3) to further increase enrichment;    -   5) exponential PCR amplification of the enriched product; and    -   6) analysis of the data obtained in step f), comprising grouping        reads by the primary barcode (R) and considering the number of        primary barcodes R, building consensus sequences, considering        the number of the secondary barcodes (S) and R for each        grouping, and calling for mutants.

Exemplary mutant enrichment of primary and secondary barcoded sequencescan be performed as described or by any other method.

In another exemplary embodiment of the present disclosure, a method canbe provided for sequencing a genomic DNA, comprising:

(a) digesting the genomic DNA at a 3′ end of a region of interest toobtain a digested genomic DNA;

(b) performing a single cycle of linear amplification of the digestedgenomic DNA with a primary barcoded adapter comprising a primarybarcode, where a regular DNA polymerase may be used in the performanceof the single cycle for sequencing a DNA, and where a reversetranscriptase may be used in procedure (b) for sequencing a RNA;

(c) removing unused primary barcode by digestion with a single-strandedDNA exonuclease to obtain a first product;

(d) performing N cycles linear amplification of the first product usinga plurality of secondary barcoded adapters, each of the plurality ofsecondary barcoded adapters comprising a secondary barcode, to obtainprimary and secondary barcoded molecules;

(e) performing exponential PCR of the primary and secondary barcodedmolecules to obtain a library comprising exponential PCR amplifiedprimary and secondary barcoded molecules;

(f) generating single stranded DNA from the exponential PCR amplifiedprimary and secondary barcoded molecules;

(g) annealing the single stranded primary and secondary barcodedmolecules to a wild-type sequence that does not overlap a degenerateregion of the library to form annealed molecules each having an annealedregion;

(h) cleaving homoduplex molecules with duplex specific nuclease toobtain an enriched product;

(i) repetition of procedure (g) and (h) to further enrich the library;

(j) amplifying the enriched product to obtain an amplified product;

(k) sequencing the amplified product; and

(l) analyzing the data obtained in procedure (k).

According to further exemplary embodiments, systems, kits andcomputer-accessible medium can be provided to perform the exemplaryprocedures described herein using computer software and hardwareprocessors.

These and other objects, features and advantages of the exemplaryembodiments of the present disclosure will become apparent upon readingthe following detailed description of the exemplary embodiments of thepresent disclosure, when taken in conjunction with the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Further objects, features and advantages of the present disclosure willbecome apparent from the following detailed description taken inconjunction with the accompanying Figures showing illustrativeembodiments of the present disclosure, in which:

FIG. 1 is an illustration of an exemplary comparison of maximum depthenrichment sequencing (MDES) with maximum-depth sequencing (MDS)according to an exemplary embodiment of the present disclosure;

FIG. 2 is an illustration of secondary barcoding preserving the abilityto identify polymerase errors following mutant enrichment, according toan exemplary embodiment of the present disclosure;

FIG. 3 is an illustration of a high accuracy of an exemplary embodimentof the present disclosure in genomic DNA collected from three patientbuffy coat samples when compared to MDS. Accuracy is increased as thesecondary barcode threshold “S” is increased. “e2” and “e3” signify twoand three repetitions of the mutation enrichment step, respectively; and

FIG. 4 is an illustration demonstrating a two orders of magnitudeenrichment of mutation informing reads following an exemplary embodimentof the present disclosure in genomic DNA collected from three patientbuffy coat samples. “e2” and “e3” signify two and three repetitions ofthe mutation enrichment step, respectively.

Throughout the drawings, the same reference numerals and characters,unless otherwise stated, are used to denote like features, elements,components or portions of the illustrated embodiments. Moreover, whilethe present disclosure will now be described in detail with reference tothe figures, it is done so in connection with the illustrativeembodiments and is not limited by the certain exemplary embodimentsillustrated in the figures and the appended claims.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Maximum depth enrichment sequencing (MDES) as described herein accordingto certain exemplary embodiments can be broadly applicable as anextension to any sequencing method and/or procedure that utilizesbarcoding such as, e.g., MDS, and any other derivatives. As an example,the adaptation of this exemplary procedure to MDS is described herein,because it is the current most sensitive and specific technology. A moredetailed discussion of MDS can be found in, e.g., Jee et al., Rates andmechanisms of bacterial mutagenesis from maximum-depth sequencing,Nature 534, 693-6 (2016) and U.S. Patent Application Publication No.2017/0016062, the disclosure of which are hereby incorporated byreference in their entireties.

FIG. 1 shows an illustration of a comparison of maximum depth enrichmentsequencing (MDES) with maximum-depth sequencing (MDS) according to anexemplary embodiments of the present disclosure. For example, MDSinvolves (a) digestion of genomic DNA at the 3′ end of a region ofinterest, (b) linear amplification of the digested genomic DNA with abarcoded adapter to directly barcode the original molecule, (c) removalof unused barcodes by digestion with an single-stranded DNA exonuclease,(d) N cycles linear amplification of this product to produce severalcopies of the original molecule, (e) exponential polymerase chainreaction (PCR) of the linear amplification product, and (f) sequencingof the final library. The barcode can be denoted from the unmodified MDSprotocol as the primary barcode (B1).

In the exemplary adaptation of MDS to MDES, a secondary barcode (B2)should be added during (d) N cycles of linear amplification such thateach primary barcode family has N subfamilies, each labeled by a uniquesecondary barcode. Following this, unused secondary barcodes should alsobe removed. Because linear amplification produces single strandedproducts, bead selection or column cleanup should be used instead of asingle-strand DNA exonuclease. Alternatively or in addition, a singleround of PCR can be performed with short primers targeting the end ofthe single strand linear products before a second round of single-strandDNA digestion. The exemplary purpose of these secondary barcodes isdiscussed in more detail herein.

Following (e) exponential PCR of these primary and secondary barcodedmolecules, single stranded DNA can be generated by any suitable method.One exemplary method includes biotinylation and immobilization of onestrand of the library, and then removal of the complementary strand withalkaline conditions. Single stranded primary and secondary barcodedmolecules can then be annealed to a wild-type reverse complementsequence that does not overlap the degenerate regions of the library toform homo and heteroduplexes that correspond to wild-type and mutantsequences respectively. Subsequently, a duplex specific nuclease can beused to digest wild-type homoduplexes, effectively enriching mutantsequences by negative selection which are then amplified for sequencing.

In this exemplary case, duplex specific nuclease isolated from theKamchatka crab is used for enrichment. The exemplary mutant enrichmentstep is performed in a similar manner to the method: NaME-PrO—ablationof WT sequences (Song, C. Elimination of unaltered DNA in mixed clinicalsamples via nuclease-assisted minor-allele enrichment. Nucleic AcidsRes. 44(19): e146 (2016)). However, the exemplary approach applies theenrichment step to single-stranded primary and secondary barcodedmolecules while NaME-PrO applies enrichment directly to unmanipulatedgenomic DNA. Alternatively or in addition, the exemplary mutantenrichment of primary and secondary barcoded molecules can be performedusing positive selection with a mismatch nuclease such as CELII or withany other methods.

Following sequencing, the exemplary analysis workflow is like MDS inthat it can include grouping primary barcodes into families and callingfor a consensus sequence. Typically, the number of reads for eachprimary barcode is recorded as “R”, filtering is done to find families,for example, with R≥3, a consensus sequence is found, and mutations areidentified. It is possible to refer to MDS documentation for details inthe selection of R≥3 and the calculated error rate. MDS documentationincludes, and is not limited to, U.S. Patent Publication No.2017/0016062; European Patent Publication No. 2107124; The TRACERxconsortium et al. Phylogenetic ctDNA analysis depicts early-stage lungcancer evolution. Nature 545, 446-451 (2017); and Jee, J. et al. Ratesand mechanisms of bacterial mutagenesis from maximum-depth sequencing.Nature 534, 693-6 (2016), the disclosures of which are incorporatedherein by reference in their entirety.

For MDES, notable additions to the analysis workflow can include, e.g.,retaining the number of unique secondary barcodes (denoted as “S”) ineach primary barcode family, modified calling for consensus sequences,filtering for true mutants, and estimating the mutant frequency.

FIG. 2 shows an illustration that secondary barcoding preserves theability to identify polymerase errors following mutant enrichment,according to an exemplary embodiment of the present disclosure. Theexemplary use of secondary barcoding can be important for correctlyidentifying mutants in the method according to certain exemplaryembodiments of the present disclosure. This can be because any methodthat aims to enrich for mutants after barcoding and amplification losesthe ability to call for a consensus sequence. For example, it ispossible to utilize a true wild-type primary barcode family where apolymerase error is made in a single copy during (d) N cycles of linearamplification. Assuming proportional exponential amplification, 1/N ofthe family would contain this error. With MDS, a majority vote strategywhen calling consensus would properly call this as a wild-type sequence.However, if mutant sequences are enriched and selected for, thewild-type sequences are lost and MDES would erroneously call this amutant sequence. By counting the number of unique secondary barcodes ina primary barcode family, the false positive error rate can becalculated to be EAS, with E being the estimated probability of apolymerase error occurring per nucleotide. As E is less then 1E-6 formodern DNA polymerases, this value clearly decreases to insignificantlevels with, for example, S≥2. In certain exemplary embodiments, thethreshold for S can be increased to further decrease the false positiveerror rate.

FIG. 3 is an illustration of a high accuracy of an exemplary embodimentof the present disclosure when compared to MDS. MDS and MDES librarieswere prepared from genomic DNA isolated from three patient buffy coatsamples in a way such that consensus sequences sharing the same primarybarcode would be identical between each method. Because MDS is wellestablished as a highly accurate method, it is used as ground truth whenbenchmarking the enriched method. As expected, accuracy of the enrichedmethod is increased as the secondary barcode threshold “S” is increasedto ≥2. The labels “e2” and “e3” signify repetition of the mutantenrichment step two and three times respectively.

FIG. 4 is an illustration demonstrating a two orders of magnitudeenrichment of mutation informing reads in an exemplary embodiment of thepresent disclosure when compared to MDS with R≥3 and S≥2. Thiscorresponds to a near 100 fold decrease in sequencing space and costswhen using MDES with the same accuracy and sensitivity MDS affords, or a100 fold increase in sensitivity if the amount of sequencing space iskept constant.

Thus, the exemplary strategy for analysis of MDES data is then to groupfamilies by primary barcode, vote for consensus sequences, and record Rand S. After selecting for sequences with satisfactory R≥3 and S≥2,mutants can be confidently identified to the same accuracy of MDS withgreater than 100 fold increase in read efficiency.

In summary, the methods and procedures according to an exemplaryembodiment according to the present disclosure can address the variousdeficiencies in the prior methods and systems, and can comprise, e.g.:secondary barcoding of primary barcoded molecules in order to preserveerror correction throughout mutant enrichment, removal of unusedsecondary barcodes, annealing of barcoded libraries to known wild-typesequences to form homo or heteroduplexes, duplex specific nucleasedigestion of the aforementioned primary and secondary barcoded duplexes,amplification of the remaining mutant products, and sequence analysisusing a strategy in which the number of unique secondary barcodes perprimary barcode are counted and taken into consideration. Theseexemplary features according to various exemplary embodiments of thepresent disclosure may be applied to other barcoding methods withminimal adjustment.

To reiterate the exemplary improvements upon existing technologiesdescribed herein, MDES increases the sensitivity and efficiency of raremutant detection far beyond any current methods. Certain competingtechnologies include other barcoding methods such as duplex sequencingand MDS, but these conventional technologies are prohibitively expensivefor widespread adoption and are likely only reasonably sensitive to5E-5. Certain disadvantages of other NGS error-correction methods arediscussed in, e.g., Jee et al., Rates and mechanisms of bacterialmutagenesis from maximum-depth sequencing. Nature 534, 693-6 (2016).

MDES according to various exemplary embodiments of the presentdisclosure can be used to make any amplicon barcoding method moresensitive and more affordable. One of certain important applications isin cancer prognosis and screening. As described herein, MDES accordingto various exemplary embodiments of the present disclosure can decreasecosts more than 100-fold, allowing for affordable screening of thousandsof ROI. This exemplary technology can provide early detection ofinitiating mutations, relapse, and resistance in cancer from a singleblood draw. Additional exemplary applications are described in U.S.Patent Application Publication No. 2017/0016062, e.g., paragraph [0033],the entire disclosure of which is incorporated herein by reference.

The foregoing merely illustrates the principles of the disclosure.Various modifications and alterations to the described embodiments willbe apparent to those skilled in the art in view of the teachings herein.It will thus be appreciated that those skilled in the art will be ableto de-vise numerous systems, arrangements, and procedures which,although not explicitly shown or described herein, embody the principlesof the disclosure and can be thus within the spirit and scope of thedisclosure. Various different exemplary embodiments can be used togetherwith one another, as well as interchangeably therewith, as should beunderstood by those having ordinary skill in the art. In addition,certain terms used in the present disclosure, including thespecification, drawings and claims thereof, can be used synonymously incertain instances, including, but not limited to, for example, data andinformation. It should be understood that, while these words, and/orother words that can be synonymous to one another, can be usedsynonymously herein, that there can be instances when such words can beintended to not be used synonymously. Further, to the extent that theprior art knowledge has not been explicitly incorporated by referenceherein above, it is explicitly incorporated herein in its entirety. Allpublications referenced are incorporated herein by reference in theirentireties.

EXEMPLARY REFERENCES

The following references are hereby incorporated by reference, in theirentireties:

-   1. U.S. Patent Publication No. 2017/0016062-   2. European Patent Publication No. 2107124-   3. The TRACERx consortium et al. Phylogenetic ctDNA analysis depicts    early-stage lung cancer evolution. Nature 545, 446-451 (2017)-   4. Jee, J. et al. Rates and mechanisms of bacterial mutagenesis from    maximum-depth sequencing. Nature 534, 693-6 (2016)-   5. Song, C. Elimination of unaltered DNA in mixed clinical samples    via nuclease-assisted minor-allele enrichment. Nucleic Acids Res.    44(19): e146 (2016).)

What is claimed is:
 1. A method for sequencing a nucleic acid,comprising: (a) digesting the nucleic acid that includes an originalmolecule at a 3′ end of a region of interest to obtain a digestednucleic acid; (b) performing a linear amplification of the digestednucleic acid with a primary barcoded adapter comprising a primarybarcode to directly barcode the original molecule; (c) removing unusedprimary barcodes by a digestion with a single-stranded DNA exonucleaseto obtain a first product; (d) performing N cycles of linearamplification of the first product using a plurality of secondarybarcoded adapters, each of the plurality of secondary barcoded adapterscomprising a secondary barcode; (e) removing unused secondary barcodesto produce a second product comprising a first library of multiplecopies of the original molecule, wherein the N cycles of linearamplification add the secondary barcode such that each of the primarybarcodes has N secondary barcodes; (f) after procedure (e), performing amutant enrichment on the second product to obtain a second library; and(g) sequencing the second library.
 2. The method of claim 1, furthercomprising: performing a data analysis of the sequenced second library,wherein the performance of the data analysis includes grouping reads bythe primary barcode (R) into a plurality of groups, filtering forsufficient R, building consensus sequences, considering the number ofthe secondary barcodes (S) and R for each group, and calling formutants.
 3. The method of claim 1, wherein the removal of the unusedsecondary barcodes is performed by (i) an additional one cycle of PCRwith a reverse amplifier to protect single-stranded linear amplificationproducts followed by digestion with a single-stranded DNA exonuclease,or (ii) a size selection using beads, gel or column purification.
 4. Themethod of claim 1, wherein procedure (a) when the nucleic acid is DNAcomprises digesting the DNA at the 3′ end of the region of interest withan enzyme.
 5. The method of claim 4, wherein the enzyme is a restrictionenzyme or CRISPR/cas9.
 6. The method of claim 1, wherein procedure (a),when the nucleic acid is RNA, comprises digesting the RNA at the 3′ endof the region of interest with an enzyme.
 7. The method of claim 6,wherein the enzyme is at least one of RNAse H or eukaryotic Argonaute.8. A method for sequencing a genoprimarymic DNA, comprising: (a)digesting the genomic DNA at a 3′ end of a region of interest to obtaina digested genomic DNA; (b) performing a single cycle of a linearamplification of the digested genomic DNA with a primary barcodedadapter comprising a primary barcode; (c) removing an unused primarybarcode by digestion with a single-stranded DNA exonuclease to obtain afirst product; (d) performing an N cycles linear amplification of thefirst product using a plurality of secondary barcoded adapters, each ofthe plurality of secondary barcoded adapters comprising a secondarybarcode, followed by a removal of unused secondary barcodes, to obtainprimary and secondary barcoded molecules such that each primary barcodehas N number of the secondary barcodes; (e) performing exponentialpolymerase chain reaction (PCR) of the primary and secondary barcodedmolecules to obtain a library comprising exponential PCR amplifiedprimary and secondary barcoded molecules; (f) generating a singlestranded DNA from the library; (g) annealing the single stranded primaryand secondary barcoded molecules to a wild-type sequence that does notoverlap a degenerate region of the library to form annealed moleculeseach having an annealed region; (h) cleaving the annealed molecules witha duplex specific nuclease to obtain an enriched product; (i) repeatingprocedures (g) and (h) to further enrich the enriched product; (j)amplifying the enriched product with exponential PCR to obtain anamplified enriched product suitable for sequencing; and (k) sequencingthe amplified enriched product.
 9. The method of claim 8, wherein theduplex specific nuclease in procedure (h) cleaves the homoduplexes,thereby enriching the library for mutants by ablating wild-typesequences.
 10. The method of claim 8, wherein the duplex specificnuclease is isolated from a Kamchatka crab.
 11. A kit for performing themethod of claim 1, comprising adapter primary barcode primers, adaptersecondary barcode primers, reverse complement primers, forward adapteramplifier primers, adapter reverse primers and reverse adapter amplifierprimers for a region of interest in the genome of an organism,optionally a buffer, and optionally an enzyme.
 12. A kit for performingthe method of claim 8, comprising adapter primary barcode primers,adapter secondary barcode primers, reverse complement primers, forwardadapter amplifier primers, adapter reverse primers and reverse adapteramplifier primers for a region of interest in the genome of an organism,optionally a buffer, and optionally an enzyme.
 13. Computer-accessiblemedium with software thereon which, when executed by a computerprocessor, configures the computer processor to effectuate theperformance of the method of claim
 2. 14. A method for sequencing anRNA, comprising: (a) digesting the RNA at the 3′ end of a region ofinterest to obtain a digested nucleic acid; (b) performing a linearamplification of the digested RNA with a primary barcoded adaptercomprising a primary barcode to directly barcode the original molecule;(c) removing unused primary barcodes by the digestion with asingle-stranded DNA exonuclease to obtain a product; (d) exponential PCRamplification of the product obtained in procedure (c) to obtain aproduct; and (e) sequencing the product obtained in procedure (d). 15.The method of claim 14, further comprising: performing a data analysisof the sequenced data from procedure (e) using a computer processor,wherein the performance of the data analysis includes grouping reads bythe primary barcode (R), filtering for sufficient R, building consensussequences, and calling for mutants.
 16. The method of claim 14, whereina reverse transcriptase is used in (b).
 17. The method of claim 14,wherein RNAse H or eukaryotic Argonaute is used in digesting the RNA in(a).
 18. A kit for performing the method of claim 14, comprising adapterprimary barcode primers, forward adapter amplifier primers, adapterreverse primers and reverse adapter amplifier primers for a region ofinterest in the genome of an organism, optionally a buffer, andoptionally an enzyme.
 19. Computer-accessible medium with softwarethereon which, when executed by a computer processor, configures thecomputer processor to effectuate the performance of the method of claim15.